Heat shock proteins (HSPs) (such as hsp70, hsp90, calreticulin families) are abundant soluble intracellular proteins. They possess powerful immunological properties, that include potentiation of innate and adaptive components of T cell immune response, as well as its down-regulation. The immunological activities of HSPs are dependent upon the interaction of HSPs with antigen presenting cells (APCs). Based on this dependence, the presence of an HSP receptor was envisaged in 1994. Such a receptor, CD91, was structurally identified and characterized and its functional activity demonstrated, in 2000. In this application, we aim to (i) assess other reported HSP receptors and identify new HSP receptors on human dendritic cells (DCs), (ii) define HSP-CD91 interaction in structural terms, (iii) test functional activity of CD91 in vivo in mediating cross-presentation antigens, and (iv) test the adjuvanticity of other CD91 ligands for immunotherapy. Specifically, we propose to study: Aim 1: Assessment and identification of HSP receptors on murine and human DCs. (a) Assessment of LOX-1 and other reported HSP receptors with respect to their relative roles vis-a-vis CD91, in cross-presentation of HSP-chaperoned peptides;(b) Generation of monoclonal antibodies to human HSP receptors, characterized by their ability to inhibit cross-presentation of HSP-chaperoned peptides. Aim 2: Structural analysis of HSP-CD91 interaction. (a) Definition of the hsp90/gp96/hsp70 domains involved in interaction with CD91, and if necessary, LOX1 ;(b) Definition of the CD91 domain(s) involved in interaction with hsp90 and gp96. Aim 3: Role of CD91 in cross-presentation and cross-priming. (a) Test influence of anti-CD91 antibodies on cross-presentation in vitro and on cross-priming in vivo, using HSP peptide complexes or whole cells as immunogens; (b) Test the influence of CD91 ligands alpha2 macroglobulin (alpha2M) and RAP on the same; (c) Test the influence of excess peptide-free HSPs on the same; (d) Construct mice expressing dominant negative forms of CD91 and test priming and cross-priming of T cell responses in such mice. Aim 4: Mechanism of immunogenicity of alpha2M and its application to cancer immunotherapy (a) Examine the mechanism of immunogenicity and anti-tumor reactivity of alpha2M-peptide complexes; (b) Test complexes of antigenic peptides with alpha2M in cross-presentation in vitro and for therapy of pre-existing cancers.